Galectin‐3 negatively regulates the frequency and function of CD4+CD25+Foxp3+ regulatory T cells and influences the course of Leishmania major infection

Galectin‐3, an endogenous glycan‐binding protein, plays essential roles during microbial infection by modulating innate and adaptive immunity. However, the role of galectin‐3 within the CD4⁺CD25⁺Foxp3⁺ T regulatory (T_REG) cell compartment has not yet been explored. Here, we found, in a model of Leishmania major infection, that galectin‐3 deficiency increases the frequency of peripheral T_REG cells both in draining lymph nodes (LNs) and sites of infection. These observations correlated with an increased severity of the disease, as shown by increased footpad swelling and parasite burden. Galectin‐3‐deficient (Lgals3^?/?) T_REG cells displayed higher CD103 expression, showed greater suppressive capacity, and synthesized higher amounts of IL‐10 compared with their wild‐type (WT) counterpart. Furthermore, both T_REG cells and T effector (T_EFF) cells from Lgals3^?/? mice showed higher expression of Notch1 and the Notch target gene Hes‐1. Interestingly, Notch signaling components were also altered in both T_REG and T_EFF cells from uninfected Lgals3^?/? mice. Thus, endogenous galectin‐3 regulates the frequency and function of CD4⁺CD25⁺Foxp3⁺ T_REG cells and alters the course of L. major infection.     

Deletion of Galectin‐3 attenuates acute pancreatitis in mice by affecting activation of innate inflammatory cells

Acute pancreatitis is characterized by autodigestion of pancreatic cells followed by acute inflammation leading to pathology and death. In experimental acute pancreatitis, pancreatic acinar cells and infiltrating macrophages express Galectin‐3 but its role in pathology of this disease is unknown. Therefore, we studied its role using Galectin‐3 deficient mice. Deletion of Galectin‐3 prolonged the survival of mice, led to attenuation of histopathology, and decreased infiltration of mononuclear cells and neutrophils that express TLR‐4, in particular, pro‐inflammatory N1 neutrophils. Galectin‐3 and TLR‐4 are also colocalized on infiltrating cells. Lack of Galectin‐3 reduced expression of pro‐inflammatory TNF‐α and IL‐1β in F4/80⁺CD11c‐ and CD11c⁺F4/80^? cells. Thus, deletion of Galectin‐3 ameliorates acute pancreatitis by attenuating early influx of neutrophils and inflammatory mononuclear cells of innate immunity. These findings provide the basis to consider Galectin‐3 as a therapeutic target in acute pancreatitis.     

Galectin‐9 expands immunosuppressive macrophages to ameliorate T‐cell‐mediated lung inflammation

Galectin‐9 (Gal‐9) plays pivotal roles in the modulation of innate and adaptive immunity to suppress T‐cell‐mediated autoimmune models. However, it remains unclear if Gal‐9 plays a suppressive role for T‐cell function in non‐autoimmune disease models. We assessed the effects of Gal‐9 on experimental hypersensitivity pneumonitis induced by Trichosporon asahii. When Gal‐9 was given subcutaneously to C57BL/6 mice at the time of challenge with T. asahii, it significantly suppressed T. asahii‐induced lung inflammation, as the levels of IL‐1, IL‐6, IFN‐γ, and IL‐17 were significantly reduced in the BALF of Gal‐9‐treated mice. Moreover, co‐culture of anti‐CD3‐stimulated CD4 T cells with BALF cells harvested from Gal‐9‐treated mice on day 1 resulted in diminished CD4 T‐cell proliferation and decreased levels of IFN‐γ and IL‐17. CD11b⁺Ly‐6C^highF4/80⁺ BALF M? expanded by Gal‐9 were responsible for the suppression. We further found in vitro that Gal‐9, only in the presence of T. asahii, expands CD11b⁺Ly‐6C^highF4/80⁺ cells from BM cells, and the cells suppress T‐cell proliferation and IFN‐γ and IL‐17 production. The present results indicate that Gal‐9 expands immunosuppressive CD11b⁺Ly‐6C^high M? to ameliorate Th1/Th17 cell‐mediated hypersensitivity pneumonitis.     

The Role of T cell Immunoglobulin and Mucin Domain‐3 in Immune Thrombocytopenia

T cell immunoglobulin and mucin domain‐3 (TIM‐3), originally identified as a T helper (Th) 1‐specific type I membrane protein, plays a vital role in Th1 immunity and tolerance induction through interaction with its ligand, galectin‐9. The binding of TIM‐3 by galectin‐9 serves to downregulate Th1 responses. Moreover, the regulatory function of TIM‐3 has been extended to other cells, such as Th17 cells, CD4⁺CD25⁺ regulatory T cells (Tregs), CD8⁺ T cells and certain innate immune cells. Previous studies have acknowledged that the TIM‐3 pathway is involved in the pathogenesis of several human autoimmune diseases, such as systemic lupus erythematous, rheumatoid arthritis and aplastic anaemia. Moreover, genetic data suggest a role for TIM‐3 in human autoimmune diseases. However, in immune thrombocytopenia (ITP), a common Th1‐ and possibly Th17‐biased autoimmune disorder, the role of TIM‐3 has not been explored. Recently, our data have demonstrated that TIM‐3 expression is reduced in ITP patients, and we have found a potential link between ITP and the TIM‐3 pathway. In this article, we discuss and speculate on the role of the TIM‐3 pathway in ITP.     

CD169 identifies an anti‐tumour macrophage subpopulation in human hepatocellular carcinoma

Macrophages are a major component of most solid tumours and can exert both anti‐ and pro‐tumourigenic functions. Although the immunosuppressive/pro‐tumour roles of macrophages have been widely examined, significantly less is known about macrophage subpopulations that have potential anti‐tumour properties in humans. In the present study, a population of CD169⁺ macrophages with relatively high expression levels of HLA‐DR and CD86 was identified in human hepatocellular carcinoma tissues. The frequency of CD169‐expressing macrophages within cancer nests was significantly lower than that found in paired non‐tumour areas. In vitro experiments revealed that in the presence of anti‐CD3 stimulation, CD169⁺ macrophages could significantly enhance the proliferation, cytotoxicity, and cytokine production capacity of CD8⁺ T cells in a CD169 molecule‐dependent manner. Autocrine TGF‐β produced by tumour‐stimulated macrophages was involved in the down‐regulation of CD169 expression on these cells. Moreover, the accumulation of CD169⁺ macrophages in tumour tissues was negatively associated with disease progression and predicted favourable survival in hepatocellular carcinoma patients, which was in contrast to the trend observed for total CD68⁺ macrophages. Therefore, CD169 might act as a co‐stimulatory molecule for cytotoxic T‐cell activation, and could define a population of tumour‐infiltrating macrophages with potential anti‐tumour properties in human hepatocellular carcinoma tissues. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Proinflammatory stimuli induce galectin‐9 in human mesenchymal stromal cells to suppress T‐cell proliferation

Human multipotent mesenchymal stromal cells (MSCs) are clinically applied to treat autoimmune diseases and graft‐versus‐host disease due to their immunomodulatory properties. Several molecules have been identified to mediate these effects, including constitutively expressed galectin‐1. However, there are indications in the literature that MSCs exert enhanced immunosuppressive functions after interaction with an inflammatory environment. Therefore, we analyzed how inflammatory stimuli influence the expression of the galectin network in MSCs and functionally tested the relevance for the immunomodulatory effects of MSCs. We found that galectin‐9 was strongly induced in MSCs upon interaction with activated PBMCs. Proinflammatory cytokines, such as interferon‐gamma (IFN‐γ) and tumor necrosis factor‐alpha (TNF‐α), and also ligands of the Toll‐like receptors (TLRs) TLR2, TLR3, and TLR4 elicited similar induction of galectin‐9 in activated PBMCs. Galectin‐9 was not only upregulated intracellularly, but also released by MSCs in significant amounts into the supernatant after exposure to proinflammatory stimuli. In proliferation assays, MSCs with a galectin‐9 knockdown lost a significant portion of their antiproliferative effects on T cells. In conclusion, we found that unlike constitutively expressed galectin‐1, galectin‐9 is induced by several proinflammatory stimuli and released by MSCs. Thus, galectin‐9 contributes to the inducible immunomodulatory functions of MSCs.     

Psoriasis in humans is associated with down-regulation of galectins in dendritic cells

We have investigated the expression and role of galectin‐1 and other galectins in psoriasis and in the Th1/Th17 effector and dendritic cell responses associated with this chronic inflammatory skin condition. To determine differences between psoriasis patients and healthy donors, expression of galectins was analysed by RT‐PCR in skin samples and on epidermal and peripheral blood dendritic cells by immunofluorescence and flow cytometry. In the skin of healthy donors, galectin‐1, ‐3 and ‐9 were expressed in a high proportion of Langerhans cells. Also, galectins were differentially expressed in peripheral blood dendritic cell subsets; galectin‐1 and galectin‐9 were highly expressed in peripheral myeloid dendritic cells compared with plasmacytoid dendritic cells. We found that non‐lesional as well as lesional skin samples from psoriasis patients had low levels of galectin‐1 at the mRNA and protein levels, in parallel with low levels of IL‐10 mRNA compared with skin from healthy patients. However, only lesional skin samples expressed high levels of Th1/Th17 cytokines. The analysis of galectin‐1 expression showed that this protein was down‐regulated in Langerhans cells and dermal dendritic cells as well as in peripheral blood CD11c⁺ DCs from psoriasis patients. Expression of galectin‐1 correlated with IL‐17 and IL‐10 expression and with the psoriasis area and index activity. Addition of galectin‐1 to co‐cultures of human monocyte‐derived dendritic cells with autologous T lymphocytes from psoriasis patients attenuated the Th1 response. Conversely, blockade of galectin binding increased IFNγ production and inhibited IL‐10 secretion in co‐cultures of monocyte‐derived dendritic cells with CD4⁺ T cells. Our results suggest a model in which galectin‐1 down‐regulation contributes to the exacerbation of the Th1/Th17 effector response in psoriasis patients. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

CD4+ T‐cell‐mediated anti‐tumor immunity can be uncoupled from autoimmunity via the STAT4/STAT6 signaling axis

Previous reports have suggested that autoimmune sequelae may be an unavoidable consequence of successful immunization against tumor‐associated antigens, which are typically non‐mutated self‐antigens. Using a melanoma model, we demonstrated that CD4⁺ T‐cell‐mediated anti‐tumor immunity and autoimmunity could be separated by modulating the STAT4/STAT6 signaling axis. Our results have revealed an unexpected dichotomy in the effector phase following cancer vaccination where anti‐tumor immunity is mediated via a STAT6 and IL‐4‐dependent pathway, whereas autoimmune pathology is mediated via STAT4 through a mechanism that relies partially on IFN‐γ. Our results offer a possibility to elicit specific anti‐tumor responses without triggering unwanted tissue autoimmune diseases.     

In vivo depletion of DC impairs the anti‐tumor effect of agonistic anti‐CD137 mAb

Anti‐CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti‐tumor effect involves co‐stimulation of tumor‐specific CD8⁺ T cells. Whether antigen cross‐presenting DC are required for the efficacy of anti‐CD137 mAb treatment has never been examined. Here we show that the administration of anti‐CD137 mAb eradicates EG7‐OVA tumors by a strictly CD8β⁺ T‐cell‐dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross‐presentation revealed that CD11c⁺ cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor‐draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti‐OVA CTL induction. Using CD11c diphtheria toxin receptor‐green fluorescent protein→C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross‐presentation in CTL induction against OVA_257–264 epitope and in the antitumor efficacy induced by anti‐CD137 mAb.     

Interleukin‐33 and alveolar macrophages contribute to the mechanisms underlying the exacerbation of IgE‐mediated airway inflammation and remodelling in mice

Allergen‐specific IgE has long been regarded as a major molecular component of allergic asthma. Additionally, there is increasing evidence of the important roles of interleukin‐33 (IL‐33) in the disease. Here, we show that IL‐33 and alveolar macrophages play essential roles in the exacerbation of IgE‐mediated airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA)‐specific IgE monoclonal antibody (mAb) were challenged with OVA seven times intratracheally. The seventh challenge exacerbated airway inflammation and remodelling compared with the fourth challenge; furthermore, markedly increased expression of IL‐33 in the lungs was observed at the fourth and seventh challenges. When anti‐IL‐33 or anti‐ST2 antibody was administered during the fourth to seventh challenge, airway inflammation and remodelling were significantly inhibited at the seventh challenge. Because increases of IL‐33⁺ and ST2⁺ alveolar macrophages and ST2⁺ CD4⁺ T cells in the lungs were observed at the fourth challenge, the roles of macrophages and CD4⁺ cells were investigated. Depletion of macrophages by 2‐chloroadenosine during the fourth to seventh challenge suppressed airway inflammation and remodelling, and IL‐33 production in the lung at the seventh challenge; additionally, anti‐CD4 mAb inhibited airway inflammation, but not airway remodelling and IL‐33 production. Meanwhile, treatment with 2‐chloroadenosine or anti‐CD4 mAb decreased IL‐33‐induced airway inflammation in normal mice; airway remodelling was repressed only by 2‐chloroadenosine. These results illustrate that macrophage‐derived IL‐33 contributes to the exacerbation of IgE‐mediated airway inflammation by mechanisms associated with macrophages and CD4⁺ cells, and airway remodelling through the activation of macrophages.     

CD8‐β ADP‐ribosylation affects CD8+ T‐cell function

The CD8αβ coreceptor is crucial for effective peptide: MHC‐I recognition by the TCR of CD8⁺ T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD⁺ to transfer ADP‐ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD⁺, ART2.2 caused ADP‐ribosylation of CD8‐β on murine CD8⁺ T cells in vitro and in vivo. Treatment with NAD⁺ prevented binding of anti‐CD8‐β mAb YTS156.7.7 but not of mAb H35–17.2, indicating that NAD⁺ caused modification of certain epitopes and not a general loss of CD8‐β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2‐deficient T cells or in the presence of inhibitory anti‐ART2.2 single‐domain antibodies. ADP‐ribosylation of CD8‐β occurred during cell isolation, particularly when cells were isolated from CD38‐deficient mice. Incubation of ART2‐expressing, but not of ART2‐deficient, OVA‐specific CD8⁺ T cells with NAD⁺ interfered with binding of OVA_257–264:MHC‐I tetramers. In line with this result, treatment of WT mice with NAD⁺ resulted in reduced CD8⁺ T‐cell mediated cytotoxicity in vivo. We propose that ADP‐ribosylation of CD8‐β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD⁺.     

Galectin‐3 is Persistently Increased in Early Rheumatoid Arthritis (RA) and Associates with Anti‐CCP Seropositivity and MRI Bone Lesions,While Early Fibrosis Markers Correlate with Disease Activity

Galectin‐3 has been suggested as a pro‐inflammatory mediator in animal arthritis and rheumatoid arthritis (RA). We aimed to study the serum level of galectin‐3 in patients with newly diagnosed RA and associations with disease profile, Magnetic resonance imaging (MRI) findings and seromarkers of synovial matrix inflammation. One hundred and sixty DMARD naïve patients newly diagnosed with RA were included (CIMESTRA study). Clinical, serological and imaging data were recorded before treatment and at 6 weeks, 3 and 12 months. Galectin‐3 and hyaluronan (HYA) were measured by ELISA (R&D and Corgenix, USA), and the N‐terminal propeptide of type III collagen (PIIINP) by radioimmunoassay (Orion Diagnostica, Finland). One hundred and nineteen, 87 and 60 blood donors served as controls for galectin‐3, HYA and PIIINP, respectively. Baseline galectin‐3 was significantly elevated in anti‐CCP positive (4.2 μg/l IQR [3.6;6.1]) patients as compared with anti‐CCP negatives (4.0 μg/l [2.6;4.9], P = 0.05) and controls (3.8 μg/l [3.0;4.8], P < 0.01). During treatment, galectin‐3 remained elevated, but increased transiently with peak values at 6 weeks. Galectin‐3 correlated with baseline smoking, anti‐CCP, and with MRI erosion score after 1 year of follow‐up. HYA and PIIINP were elevated (P < 0.001) irrespective of anti‐CCP status and correlated positively with synovitis assessed clinically and by MRI. HYA and PIIINP did not correlate with galectin‐3. These observations indicate that HYA and PIIINP mainly reflect expansive synovitis proliferation while galectin‐3 is more closely linked to autoimmunity, smoking and joint destructive processes.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

HCV‐infected hepatocytes drive CD4+CD25+Foxp3+ regulatory T‐cell development through the Tim‐3/Gal‐9 pathway

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T‐cell Ig and mucin domain protein‐3 (Tim‐3) and galectin‐9 (Gal‐9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim‐3/Gal‐9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim‐3/Gal‐9 interactions regulate HCV‐mediated Treg‐cell development, here we provide pilot data showing that HCV‐infected human hepatocytes express higher levels of Gal‐9 and TGF‐β, and upregulate Tim‐3 expression and regulatory cytokines TGF‐β/IL‐10 in co‐cultured human CD4⁺ T cells, driving conventional CD4⁺ T cells into CD25⁺Foxp3⁺ Treg cells. Additionally, recombinant Gal‐9 protein can transform TCR‐activated CD4⁺ T cells into Foxp3⁺ Treg cells in a dose‐dependent manner. Importantly, blocking Tim‐3/Gal‐9 ligations abrogates HCV‐mediated Treg‐cell induction by HCV‐infected hepatocytes, suggesting that Tim‐3/Gal‐9 interactions may regulate human Foxp3⁺ Treg‐cell development and function during HCV infection.     

RELT negatively regulates the early phase of the T‐cell response in mice

RELT (tumor necrosis factor receptor superfamily member 19‐like, TNFRSF19L) is a TNFR superfamily member that is primarily expressed in immune cells and lymphoid tissues, but whose immunological function is not well‐defined. Here, we show that RELT is expressed by naive T cells and DCs, and their activation or maturation decreases RELT expression. Using RELT knockout (RELT^?/?) mice, we demonstrate that RELT deficiency selectively promotes the homeostatic proliferation of CD4⁺ T cells but not CD8⁺ T cells, and enhances anti‐tumor CD8⁺ T‐cell responses. We also demonstrate, using an adoptive transfer model in which RELT is knocked‐out in either the transferred transgenic CD8⁺ T cells or the recipient melanoma‐bearing mice, that RELT on multiple immune cells limits the hyper‐response of tumor‐specific CD8⁺ T cells. Hyper‐responsiveness of RELT‐deficient T cells was induced by promoting their proliferation. Taken together, our findings suggest that RELT acts as a negative regulator that controls the early phase of T‐cell activation probably by promoting T‐cell apoptosis.     

Anti‐HIV‐1 antibody‐dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG

Antibodies with antibody‐dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV‐1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV‐specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV‐1 envelope gp140 and elicited high titers of anti‐gp140‐binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ‐receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose‐dependent manner. Antibody‐dependent killing was observed in the presence of anti‐HIV‐1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab’)₂ fragments. ADCC activity was primarily mediated by CD14⁺ monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte‐mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti‐HIV colostrum IgG have robust HIV‐1‐specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV‐1 infection. This could assist the development of novel Ab‐mediated approaches for prevention of HIV‐1 transmission.     

Depletion of tumor‐induced Treg prior to reconstitution rescues enhanced priming of tumor‐specific,therapeutic effector T cells in lymphopenic hosts

We reported previously that vaccination of reconstituted, lymphopenic mice resulted in a higher frequency of tumor‐specific effector T cells with therapeutic activity than vaccination of normal mice. Here, we show that lymphopenic mice reconstituted with spleen cells from tumor‐bearing mice (TBM), a situation that resembles the clinical condition, failed to generate tumor‐specific T cells with therapeutic efficacy. However, depletion of CD25⁺ Treg from the spleen cells of TBM restored tumor‐specific priming and therapeutic efficacy. Adding back TBM CD25⁺ Treg to CD25^? naïve and TBM donor T cells prior to reconstitution confirmed their suppressive role. CD25⁺ Treg from TBM prevented priming of tumor‐specific T cells since subsequent depletion of CD4⁺ T cells did not restore therapeutic efficacy. This effect may not be antigen‐specific as three histologically distinct tumors generated CD25⁺ Treg that could suppress the T‐cell immune response to a melanoma vaccine. Importantly, since ex vivo depletion of CD25⁺ Treg from TBM spleen cells prior to reconstitution and vaccination fully restored the generation of therapeutic effector T cells, even in animals with established tumor burden, we have initiated a translational clinical trial of this strategy in patients with metastatic melanoma.     

Exposure to anti‐PD‐1 causes functional differences in tumor‐infiltrating lymphocytes in rare solid tumors

The pervasive use of therapeutic antibodies targeting programmed cell death protein 1 (PD‐1) to boost anti‐tumor immunity has positioned this approach to become the standard‐of‐care for some solid tumor malignancies. However, little is known as to how blockade of PD‐1 may alter the function or phenotype of tumor‐infiltrating lymphocytes (TIL). We used our ongoing Phase II clinical trial of pembrolizumab for patients with rare solid tumors from various types (NCT02721732) with matched core biopsies taken at baseline and after initial dose of anti‐PD‐1 (15–21 days post‐dose) to elucidate this question. One fresh core needle biopsy was used to propagate TIL ex vivo to analyze phenotype and function using flow cytometry in both CD8⁺ and CD4⁺ TIL populations. An enriched CTLA‐4 expression in the CD4⁺ TIL population was observed in TIL expanded from the on‐treatment samples compared to TIL expanded from the matched baseline (n = 22, p = 0.0007) but was not observed in patients who experienced tumor regression. Impact on functionality was evaluated by measuring secretion of 65 soluble factors by expanded TIL from 26 patients at baseline and on‐treatment. The CD8⁺ TIL population demonstrated a diminished cytokine secretion profile post‐pembrolizumab. Overall, our study assesses the ramifications of one dose of anti‐PD‐1 on TIL in rare solid tumor types.